20
Biochem. Soc. Symp. 70, 243–251
(Printed in Great Britain)
© 2003 Biochemical Society
Separase regulation during
mitosis
Frank Uhlmann1
Lincoln’s Inn Fields Laboratories, Cancer Research UK, 44 Lincoln’s Inn Fields,
London WC2A 3PX, U.K.
Abstract
The final, irreversible step in the duplication and distribution of genomes
to daughter cells takes place when chromosomes split at the metaphase-toanaphase transition. A protease of the CD clan, separase (C50 family), is the
key regulator of this transition. During metaphase, cohesion between sister
chromatids is maintained by a chromosomal protein complex, cohesin.
Anaphase is triggered when separase cleaves the Scc1 subunit of cohesin at two
specific recognition sequences. As a result of this cleavage, the cohesin complex
is destroyed, allowing the spindle to pull sister chromatids into opposite halves
of the cell. Because of the final and irreversible nature of Scc1 cleavage, this
reaction is tightly controlled. Several independent mechanisms impose regulation on separase activity, as well as on the susceptibility of the cleavage target
Scc1 to cleavage by separase. This chapter provides an overview of these multiple levels of regulation.
Introduction
The DNA that comprises eukaryotic genomes is packaged into chromosomes. These must be replicated accurately to produce exact copies during
S-phase, and then distributed correctly during mitosis. Errors in distribution
lead to cells with supernumerary or missing chromosomes. The resulting aneuploidy is associated with many cancers, and is a leading cause of human birth
defects. It is crucial that the products of DNA replication, the sister chromatids, remain physically linked by sister chromatid cohesion after their
synthesis. This facilitates the repair of DNA lesions by recombination using
the sister chromatid as a template [1]. Sister cohesion is also fundamental to the
bipolar alignment of chromosomes on the metaphase spindle, as it counteracts
the pulling force of microtubules toward the spindle poles (reviewed in [2]). At
1e-mail
[email protected]
243
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F. Uhlmann
the start of anaphase, cohesion is abolished abruptly by a tightly regulated proteolytic cascade that activates the CD clan protease, separase. Separase cleaves
one of the subunits of the cohesin complex, the Scc1 subunit, thereby destroying the complex. This review focuses on the regulation of separase activity, and
on how cells ensure that cohesin cleavage occurs at the right time and place,
making possible the complete and accurate distribution of chromosomes.
Separase, the protease that cleaves cohesin
Separase, the protease responsible for cleaving cohesin at anaphase onset
[3,4], is a protein that was genetically identified and implicated in the regulation
of chromosome segregation some time ago. Separase homologues probably
exist in all eukaryotes, and mutations in the separases in Schizosaccharomyces
pombe (Cut1), Saccharomyces cerevisiae (Esp1) and Aspergillus nidulans
(BimB) have been characterized [5–7]. All prevent chromosome segregation at
anaphase without halting the continuation of the cell cycle. This leads to cells
with re-replicated chromosomes and excess spindle pole bodies, explaining the
original phenotypic description of Extra Spindle Poles (esp1) [8].
The primary defect in esp1 mutant cells only became apparent following
the discovery of cohesin [9]. During anaphase, two of cohesin’s subunits, Scc1
and Scc3, suddenly disappear from the chromosomes of wild-type cells (Figure
1) [10,11]. In esp1 mutant cells, however, these subunits fail to dissociate from
chromosomes, and sister chromatids remain paired even after they should have
separated [12]. This observation led to the hypothesis that separases are cohesin
removal factors (Figure 1). Meanwhile, the separases in a number of other
organisms had been identified by genome sequencing projects. Separases are
generally large proteins of close to 200 kDa, and only a C-terminal domain
seems to be conserved among them. This conserved ‘separase domain’ contains
the signature motif for cysteine proteases of the CD clan, the superfamily of
proteases that also includes the caspases. Separases have been assigned to family
C50. Indeed, separases purified from both budding yeast and human cells possess proteolytic activity against Scc1 [4,13]. There are two specific cleavage sites
within budding yeast Scc1 that display a characteristic consensus motif [3].
This motif has also aided the identification of the cleavage sites within Scc1
homologues in other species [9,14], the most important determinants of which
are an arginine in the P1 position and a negatively charged amino acid in the P3
position. In addition, a specific Scc1-derived peptide inhibitor has been developed against yeast separase, based on caspase inhibitors containing an activated
chloromethyl ketone or acyloxymethyl ketone that covalently binds to and
inhibits the separase active-site cysteine [4].
Securins: cellular separase inhibitors
The best known regulators of separases are the securins. First discovered
in both budding and fission yeast [15,16], securins have since also been characterized in metazoans ([17,18]; reviewed in [19]). They are functionally
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Separase regulation during mitosis
245
Metaphase
Anaphase
Separase
Cohesin:
Smc1/3
Scc1/3
Figure 1 Model for how cleavage of cohesin by separase destroys
sister chromatid cohesion at anaphase onset. Separase recognizes and
cleaves two distinct sites in the Scc1 subunit. This destroys the interactions
within the cohesin complex, leading to dissociation of the Scc1 and Scc3 subunits from chromosomes. Cohesion is lost, and the pulling force of the mitotic
spindle segregates the sister chromatids towards opposite poles.
conserved proteins, although there is little conservation of their primary amino
acid sequence. Securins bind to and inhibit separase for most of the cell cycle
[3,12], but are degraded at the onset of anaphase, thus releasing separase (Figure
2). Their degradation is triggered via ubiquitylation by the anaphase-promoting complex (APC) [15,20]. Although securins are potent separase inhibitors,
in budding yeast securin is not essential for cell cycle regulation of Scc1 cleavage, indicating that other control mechanisms exist (see below).
Yeast and human securins have recently been characterized as bona fide
protease inhibitors for separase [21,22]. Securin prevents an intramolecular
interaction between the N- and C-termini within separase. The protease active
site resides in the separase C-terminus, and the interaction with the N-terminus
may be required to induce an activating conformational change. Thus securin
may prevent separase’s own activation [21]. This also suggests a function for
the large N-terminal extensions of separases in the regulated activation of the
C-terminal protease domains. In addition, securin prevents binding of separase
to its substrate Scc1 [21].
Securins are not simply inhibitors of separase. In fission yeast and
Drosophila, the absence of securin does not lead to a prematurely active separase as one might predict, but, rather paradoxically, to an apparent lack of
separase activity. This suggests a dual role for securins: the priming of separase
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F. Uhlmann
Mad2
Cdc20
APC/C
u
u
u
u
Securin
Separase
Separase
Securin
Chk1
Figure 2 Separase regulation by securin. From late G1 until metaphase,
securin binds to and inhibits separase. At anaphase, securin is targeted for
destruction via ubiquitylation by the APC. The APC is activated by the regulatory subunit Cdc20/Fizzy. As long as chromosomes are not aligned properly on
the mitotic spindle, Mad2 prevents activation of the APC. After DNA damage,
the kinase Chk1 phosphorylates securin and prevents its destruction.
Reproduced from [50], with permission.
activity via binding, and the inhibition of separase until securin’s degradation
by the APC [15,23]. Even in budding yeast where securin is not essential, separase function is impaired in its absence [12]. Securin acts to concentrate
separase in the nucleus where Scc1 must be cleaved during anaphase [21,24].
Securin also enhances the specific proteolytic activity of separase after its own
destruction [21,25], indicating that it may act as a molecular chaperone, helping
separase to acquire its active conformation.
When human securin was identified, it was found to be the product of the
pituitary tumour-transforming gene, which is overexpressed in certain tumours
and exhibits transforming activity in NIH 3T3 cells [17]. Chromosome mis-segregation is thought to be a cause of the genetic instabilities found associated with
many cancers [26], and overexpressed securin might lead to incomplete chromosome segregation due to inhibition of separase activity during anaphase.
Securin regulation via the APC
The APC is a protein ubiquitin ligase that is essential for mitotic progression. It controls the degradation of numerous proteins in addition to securin at
this stage (reviewed in [27]). Whereas in yeast the APC exerts its effect on sister
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Separase regulation during mitosis
247
chromatid separation solely by targeting securin [12,28] (Figure 2), the activation of Xenopus and human separases may also require the APC-dependent
degradation of mitotic cyclins [29]. The APC is activated at anaphase by the
Cdc20/Fizzy protein, whose expression in turn is cell cycle regulated (see [27]).
Cell cycle-dependent phosphorylation of APC subunits is also required for the
activation of the APC complex [30,31].
APC activation is also the entry point for the Mad2-dependent checkpoint pathway that monitors the bipolar attachment of chromosomes to the
mitotic spindle (Figure 2) (reviewed in [32]). Unattached kinetochores send a
signal via Mad2 that keeps the APC inactive, potentially through the binding of
Mad2 to the APC activator Cdc20/Fizzy. Indeed, the budding yeast securin,
Pds1, had initially been identified as a protein required to prevent sister chromatid separation when the Mad2-dependent checkpoint pathway is activated
[28]. In yeast, this pathway for regulating securin destruction only becomes
essential once actual damage to spindle kinetochore attachment occurs. In contrast, in higher eukaryotes it acts during each cell cycle to ensure timely sister
chromatid separation [33–35].
Controlling the onset of anaphase based on the state of chromosome
attachment to the mitotic spindle seems to be of greatest importance. But it is
not the only control. If DNA is damaged, anaphase onset is delayed to allow
repair before sister sequences are separated from each other. In budding yeast,
DNA damage elicits a response pathway that uses two routes that act together
to prevent anaphase [36–38]. One route again acts via securin that is stabilized
in response to DNA damage through the action of the kinase Chk1. Chk1
directly phosphorylates the budding yeast securin, Pds1 [38]. In higher eukaryotic cells, the majority of sister DNA sequences become separated during
chromosome condensation in prophase. Accordingly, the DNA damage
response mainly down-regulates Cdk activity, which blocks cells from entering
prophase (see [32]).
Phosphorylation of the cleavage target
As described above, budding yeast securin is essential for the prevention of
anaphase onset in response to spindle or DNA damage. During undisturbed cell
cycle progression, however, yeast securin is entirely dispensable. Cleavage of
cohesin still occurs in a regulated fashion with unchanged kinetics [39]. Is there a
second regulator besides securin that can inhibit premature activation of separase? Probably not, since the overall separase activity in yeast cells lacking securin
no longer undergoes detectable changes during the cell cycle. Instead, regulation
occurs at the level of the separase cleavage target. Scc1 is a phosphoprotein whose
phosphorylation is crucial for its cleavage by separase (Figure 3) [4,40,41]. The
polo-like kinase Cdc5 in budding yeast is responsible for phosphorylation of
Scc1 during metaphase. Preventing Scc1 phosphorylation decreases the rate of
Scc1 cleavage in vivo. This effect is especially pronounced in the absence of
securin, possibly due to the impairment of separase activity [39]. Of several sites
phosphorylated in Scc1 by Cdc5, two phosphorylated serines lie adjacent to the
© 2003 Biochemical Society
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F. Uhlmann
Separase
Rad53/
Chk2
PLK
PO32
Separase
Cohesin
Figure 3 Regulation of Scc1 cleavage at the level of the cleavage substrate. Phosphorylation of the Scc1 subunit of cohesin by polo-like kinase
(PLK) is required for its efficient cleavage. Phosphorylation might be prevented
by Rad53/Chk2 after DNA damage. Reproduced from [50], with permission.
separase cleavage sites, and the affinity of separase for the cleavage sites is
increased dramatically by phosphorylation of these residues [39].
Separase regulation in higher eukaryotes
While total levels of separase in budding or fission yeast do not undergo
obvious changes during the cell cycle [12,42], the abundance of human separase
fluctuates. In human cells, separase levels are high during metaphase and
decline during anaphase [13]. Once activated, separase cleaves itself [22,29]. The
two cleavage products stay associated with each other, and cleavage initially
does not alter the activity of separase in cleaving Scc1 [43]. However, the C-terminal cleavage product, containing the separase active site, becomes unstable
after cleavage and is degraded (Figure 4). This leads to down-regulation of separase after anaphase, which might be necessary to allow the rapid rebinding of
cohesin to chromosomes during telophase that is observed in vertebrate cells
[13,44,45]. In budding yeast, separase remains active throughout G1-phase [3],
presumably until it is inactivated by the resynthesis of securin shortly before
the next S-phase.
A remarkable situation is found in Drosophila. Here, separase appears to
be split into two polypeptides. The protein containing the separase protease
domain is short compared with separases in other organisms. However, it
forms a tight complex with another protein, Three rows, that may correspond
to the long N-terminal extensions found in most separases [46]. It is the Three
rows protein that is cleaved by separase in anaphase, and the C-terminal Three
rows fragment is rendered unstable and disappears from cells. When a site-specific mutation was introduced into Three rows that made it uncleavable by
separase, this led to mitotic defects and other phenotypes that were probably
caused by a failure to down-regulate separase activity after anaphase [47].
Another level of regulation of Xenopus and human separase that is not
found in budding yeast is the inhibition of separase by mitotic phosphorylation. High cyclin-dependent kinase activity in Xenopus egg extracts inhibits
separase, and human separase in metaphase is inhibited by phosphorylation on
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Separase regulation during mitosis
249
arase
Sep
Separase
arase
Separase
p
Se
Figure 4 Human and Xenopus separases undergo self-cleavage during
anaphase. The cleavage products stay associated with each other, and cleavage initially does not change the protease activity of separase. The C-terminal
separase fragment, however, is rendered unstable by the cleavage, leading to
down-regulation of separase after anaphase.
a specific serine residue [29]. The phosphate has to be removed in anaphase,
when cyclin-dependent kinase activity decreases, before separase can become
active. This mechanism might, at least in vertebrates, help to regulate separase
in the absence of securin.
Conclusions
We now understand the molecular principle of how chromosome segregation is triggered at anaphase onset. A specific protease, separase, cleaves a
protein that is required to hold the chromosomes together. We are also beginning to understand how this reaction is regulated on a number of levels to
ensure that chromosomes are not separated prematurely. It may be that additional controls become important under certain conditions. For example,
calcium waves are thought to play a role in triggering anaphase, and separase
has a potential calcium-binding site [48], suggesting that separase activity might
be regulated by calcium levels. In addition, different-sized complexes of separase with securin have been detected in fission yeast cells, but their possible
roles have not yet been explored [42]. Finally, separase cleaves not only cohesin
and itself at anaphase onset, but also the kinetochore- and microtubule-associated protein Slk19 [49]. Thus separase may have multiple roles to ensure a
smooth transition from metaphase into anaphase. It would not be surprising if
more tasks are discovered for this formidable protease.
I thank all the members of my laboratory for discussions and for the contributions
that they have made to our understanding of separase regulation. Thanks are also
due to EMBO Reports for permission to reproduce some of the figures in modified
form from [50].
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